The goal of this project is to define characteristic features that can be used to identify metamorphic proteins, polypeptides that interconvert between unrelated structures in the native state. Human lymphotactin (Ltn/XCL1), an unusual member of the chemokine family, undergoes a reversible rearrangement between two distinct native state structures at a rate of ~1 s-1. We solved the NMR structure of each species and found that one state (Ltn10) corresponds to the conserved chemokine fold and activates XCR1, the specific G protein-coupled receptor (GPCR) for lymphotactin, whereas the other (Ltn40) forms a dimeric -sandwich with high affinity for glycosaminoglycans (GAG). The two conformations are equally abundant in physiological solution conditions. Lymphotactin provides a unique opportunity to address fundamental questions about this new category of proteins: How does one amino acid sequence simultaneously encode two entirely different native state structures with equal thermodynamic stability, and how did the metamorphic protein arise from a (presumably) single-fold ancestor? Our mechanistic hypothesis is that cold denaturation of one structure at physiological temperatures creates the potential for metamorphic interconversion with another marginally stable but unrelated structure. We will pursue three specific aims designed to reveal the origin of Ltn metamorphism in the context of its relatives in the chemokine family, none of which exhibit this behavior. First, we will define the role of cold denaturation in Ltn metamorphosis by mapping the folding energy landscape using NMR and fluorescence spectroscopy to monitor urea-induced unfolding of each conformational state. Next, we propose to identify the structural elements sufficient to stabilize an alternative native state in a non-metamorphic chemokine. We hypothesize that a duality code within the Ltn sequence is composed of both stabilizing and destabilizing structural elements that adjust the free energy of folding for each state so that they are equally populated under physiological conditions. Finally, we will use new computational methods for ancestral gene reconstruction to identify evolutionary nodes separating the metamorphic and non-metamorphic branches of the chemokine family. Experimentally defined structural, thermodynamic and functional profiles of ancient sequences (~100-400 million years old) will enable us to identify key amino acid changes leading to the emergence of structural metamorphism. Collectively, the proposed studies will assess the functional relevance of cold denaturation at physiological temperature as a mechanism for protein metamorphism and perform the first evolutionary analysis of a metamorphic protein. Additionally, this project will provide the first detailed analysis of thermodynamic stability for any chemokine, a protein family with direct relevance to many human diseases.